Aging is considered a state of chronic inflammation, which is believed to be underlying cause of several diseases and mortality associated with aging (19
). However, the mechanisms responsible for chronic inflammation in aging are not well understood. In addition, aging is also associated with increased reactivity to self antigens as evidenced by the presence of auto-antibodies to a variety of self antigens (20
). Here we show that DCs from aged display increased reactivity to human DNA, resulting in increased pro-inflammatory cytokine production and induction of T cell proliferation.
The immune system is normally protected from exposure to self dsDNA during apoptosis due to the rapid engulfment of apoptotic cells, and the abundance of extra- and intracellular DNases (23
). However, phagocytic cells may be exposed to cellular DNA following tissue necrosis, inflammation, or viral infection. Defective clearance of apoptotic cells would also result in an accumulation of late phase apoptotic cells. Previous study from our laboratory in humans (10
) and a recent study (22
) in mice suggest that apoptotic cell clearance is decreased with age. The late phase apoptotic cells are associated with additional proteolytic degradation of specific autoantigens, which may release endogenous danger signals like nuclear structures clustered in apoptotic blebs (chromatin and dsDNA) and proteins such as heat shock proteins (HSPs) causing maturation of dendritic cells resulting in T cell immunity to self (24
). Our results indicate that intracellular human DNA delivered via transfection agents such as lipofectamine can stimulate DCs to secrete cytokine and upregulate costimulatory molecules. This is in keeping with recent studies where intracellular mammalian DNA delivered via fugene (27
) stimulated murine bone marrow derived dendritic cells to mature and secrete IFN-α. Intracellular administration of double-stranded B-form DNA (B-DNA) also triggered antiviral responses, including production of type I interferons and chemokines (16
). Furthermore, macrophages from mouse embryos deficient in DNase II contain intracellular undigested DNA that causes lethal type I IFN production (28
). Mammalian DNA or RNA complexed with antibodies, RNA binding proteins or antimicrobial peptides are also able to activate APCs to secrete type I IFNs (29
) and have been shown to be involved in autoimmune disorders such as systemic lupus erythromatous (32
We have previously reported that DCs from aged secreted increased levels of pro-inflammatory cytokines TNF-α and IL-6 in response to TLR ligands (10
). Present results show that this enhanced pro-inflammatory cytokine secretion by DCs from aged is not restricted to TLR ligands but is also seen with self-antigens such as human DNA (). There is increased upregulation of costimulatory molecules CD80 and CD86 and enhanced secretion of IFN-α and IL-6 by DCs from aged donors in response to self-DNA. These results are further substantiated by our findings that DCs from aged also show increased response to late apoptotic cells (). Activation of DCs with apoptotic cells resulted in the secretion of TNF-α along with IFN-α and IL-6. Apoptotic cells contain other self-antigens besides DNA such as HSPs, HMGBP1 that can stimulate DCs. This may be responsible for the observed TNF-α secretion.
Activation of DCs converts them into fully functional APCs capable of priming T cell responses. We show that human DNA-activated DCs from aged primed T cells to proliferate () while the less activated DCs from young did not induce T cell proliferation (). The levels of IFN-γ in the DNA stimulated DCs from aged subjects was also significantly above (p<0.05) the unstimulated DCs while no such difference was observed in IFN-γ secretion from young subjects. Previous studies have reported that priming of T cells by DCs requires optimal level of co-stimulation as well as cytokine production (33
) and this may account for the increased proliferation of T cell observed in the aged. Though the DNA-stimulated DCs from aged display increased capacity to prime T cells compared to their young counterparts, nevertheless overall they displayed reduced level of T cell proliferation () compared to the young subjects. This supports previous report where reduced alloimmunity was observed in aged mice compared to young mice during solid organ transplant (34
). However, the reasons for this are presently unclear. We have observed the same phenomenon in other experiments and the underlying mechanisms are being investigated. One of the possible mechanisms may be the impaired formation of DC-T cell synapse in aging.
Earlier studies with intracellular DNA delivered via transfection reagents have shown that DNA signals through non-TLR receptors (16
). This is in agreement with our results in that the DNA was localized in the cytosol () and was not accessible to intracellular TLRs in the endosomes. The nucleic acid sensing TLR3 and TLR8 are found in the endosomes (35
). The two other known nucleic acid sensing TLRs, TLRs 7 and 9 are not expressed in human monocyte derived DCs and are also present in the endosomes (36
Furthermore, we did not find any difference in the expression of DAI (), one of the cytosolic DNA sensors identified recently. However, we did find activation of IRF-3 and phosphorylation of p65 unit of NF-κB signaling pathway, both of which are reported to be activated downstream of DAI (13
). IRF-3 activity was significantly higher in the DNA-treated DCs from aged compared to young (). Since IRF-3 is involved in the production of IFN-α (11
), it may explain the increased secretion of IFN-α in DCs from aged. The phosphorylation of p65 was also significantly enhanced in the DCs from aged subjects (). However, in contrast to IRF-3, the basal level of p65 activation was significantly higher in DCs from aged suggesting that DCs in aged are in an activated state compared to young counterparts. Abrogation of cytokine secretion by NFκB inhibitor () lent further support to this hypothesis. The activated state of DCs in aged subjects could be due to factors in the aged microenvironment such as increased pro-inflammatory cytokines that may cause the activation of DCs resulting in their increased reactivity to human DNA. Our previous studies had revealed no significant difference in the phenotype of DCs from aged and young (8
). Nevertheless, it was possible that DCs were not fully activated to manifest a mature phenotype but may still be primed or semi-activated. The phagocytic capacity of DCs decreases as they become activated and we have reported that DCs from aged are impaired in their capacity to phagocytose antigens (10
). Earlier studies from our laboratory have also shown that the expression of PTEN protein, which negatively regulates the PI3kinase signaling pathway, is increased in DCs from aged. This leads to increased activation of p38 MAP kinase and enhanced secretion of pro-inflammatory cytokines (10
) in response to TLR ligands. PI3kinase also regulates the activation of NF-κB pathway (37
). Increased PTEN expression in aged may thus account for the increased level of NF-κB activation in DCs. This semi-activated state of DCs alters the response of DCs to antigens resulting in the secretion of increased level of cytokines.
In summary, we show that DCs from aged are more reactive to human DNA and secrete increased level of pro-inflammatory cytokines. Increased reactivity to self may result in impaired tolerance and thus be one of the mechanisms of age-associated chronic inflammation and autoimmunity. Recent reports indicate that there is enhanced basal level of activation in of NF-κB in DCs from rheumatoid arthritis patients (38
). However, it remains to be determined whether similar mechanisms may be involved in other autoimmune diseases associated with increased pro-inflammatory cytokine production such as rheumatoid arthritis and systemic Lupus erythromatous.